OB protein compositions and methods

ABSTRACT

The present invention provides methods and compositions for treating excess weight by administering OB protein in a form for constant supply, at a dosage of less than or equal to about 1 mg protein/kg body weight/day. Compositions and methods used for production of recombinant murine and human OB protein are also provided. Compositions and methods for preparing recombinant murine methionyl OB protein and recombinant human methionyl OB protein, including DNA sequences, vectors, host cells, methods of fermentation, and methods of purification are provided herein.

FIELD OF THE INVENTION

[0001] The present invention relates to OB protein compositions andmethods for preparation and use thereof.

BACKGROUND

[0002] Although the molecular basis for obesity is largely unknown, theidentification of the “OB gene” and protein encoded by (“OB protein”)has shed some light on mechanisms the body uses to regulate body fatdeposition. Zhang et al., Nature 372: 425-432 (1994); see also, theCorrection at Nature 374: 479 (1995). The OB protein has beendemonstrated to be active in vivo in both ob/ob mutant mice (mice obesedue to a defect in the production of the OB gene product) as well as innormal, wild type mice. The biological activity manifests itself in,among other things, weight loss. To date, however, optimum conditionsfor obtaining the rapid weight loss in normal animals has not beenascertained. In fact, some studies have shown that, when administered byinjection, rather large dosages (10 mg of recombinant murine protein/kgbody weight/day) are necessary for normal mice to lose 2.6% of theirbody weight (at the end of a 32 day period). While presently uncertain,one explanation for the necessity of such large dosages is that theoptimum weight loss effects are seen predominantly when the protein isin constant circulation, a condition that may not be efficientlyachieved by injecting the protein.

SUMMARY OF THE INVENTION

[0003] The present invention stems from the observation that, ascompared to administering OB protein by injection, administering OBprotein by continuous pump infusion results in equivalent (or better)weight loss, in a shorter time, and with substantially lower dosages.The working example below demonstrates that a dose of 0.5 mg protein/kgbody weight/day, administered via implantable osmotic pump, results in aweight loss of over 4% (as compared to baseline weight). This is insubstantial contrast to other studies where similar, or less weight loss(at a comparable time point) was observed with intraperitoneal injectionat the relatively high dosage of 10 mg of protein/kg body weight/day.

[0004] Thus, one aspect of the present invention is a method of treatingexcess weight by administering OB protein in a form for constant supply,at a dosage of less than or equal to about 1 mg protein/kg bodyweight/day. The dosage of less than or equal to about 1 mgprotein/kg/day refers to dosages sufficient to result in observableweight loss. This is apparent from the present studies where a dosage of0.5 mg/kg/day was sufficient to result in observable weight loss whencontinuously administered. In studies where injection had been the modeof administration, far higher dosages were required for weight loss. Atinjection dosages of 0.1 and 1 mg/kg/day, substantially no weight losswas observed in wild type (normal) mice. For example, in one study, at acomparable time point (6th day), there was a 0.2% loss at the 1 mg/kgdose (data not shown). Minimal weight loss was observed at therelatively high 10 mg/kg/day dose. (1.9% weight loss at day 6, data notshown). Thus, the present invention provides for dosages of 1 mg/kg/dayor less when administered so that the supply of protein is continuous.

[0005] Connected with the present studies are the compositions andmethods used for production of recombinant murine and human OB protein.The first example below discloses the preparation of recombinant murineprotein, and the second example below discloses the preparation ofrecombinant human protein.

[0006] Additional aspects of the present invention, therefore, includethe below compositions and methods for preparing recombinant murinemethionyl OB protein and recombinant human methionyl OB protein,including DNA sequences, vectors, host cells, methods of fermentation,and methods of purification.

DETAILED DESCRIPTION

[0007] The present invention stems from the observation that continuousadministration of OB protein results in the need for much lower dosagesfor weight loss than those dosages required by acute daily injection. Asset forth above, a dosage of 1 mg protein/kg body weight/day or less,continuously administered, resulted in rapid weight loss. When theunderivatized protein was administered by acute injection at the 1mg/kg/day dose, almost no weight loss in wild type (normal) mice.

[0008] The OB protein may be selected from the recombinant murine andhuman methionyl proteins set forth below (SEQ. ID Nos. 2 and 4) or thoselacking a giutaminyl residue at position 28. (See Zhang et al, Nature,supra, at page 428.) The recombinant human OB gene product is, as amature protein, 146 amino acids; some of the DNAs obtained were observedto encode a protein lacking a glutamine residue at position 28. Zhang etal., Nature 372 at 428. The murine protein is substantially homologousto the human protein, particularly as a mature protein, and, further,particularly at the N-terminus. One may prepare an analog of therecombinant human protein by altering (such as substituting amino acidresidues), in the recombinant human sequence, the amino acids whichdiverge from the murine sequence. Because the recombinant human proteinhas biological activity in mice, such analog would likely be active.Proteins lacking an N-terminal methionyl residue, such as those producedby eukaryotic expression, are also available for use.

[0009] In addition, although the present working example involvedcontinuous administration via implantable pump, it is contemplated thatother modes of continuous administration may be practiced. For example,chemical derivatization may result in sustained release forms of theprotein which have the effect of continuous presence in the bloodstream, in predictable amounts. Thus, one may derivatize the aboveproteins to effectuate such continuous administration. The dosage of 1mg protein/kg body weight/day or less herein refers to the mass ofprotein, exclusive of other chemical moieties used to derivatize theprotein.

[0010] Generally, the present protein (herein the term “protein” is usedto include “peptide”, unless otherwise indicated) may be derivatized bythe attachment of one or more chemical moieties to the protein moiety.The chemically modified derivatives may be further formulated forintraarterial, intraperitoneal, intramuscular subcutaneous, intravenous,oral, nasal, pulmonary, topical or other routes of administration.Chemical modification of biologically active proteins has been found toprovide additional advantages under certain circumstances, such asincreasing the stability and circulation time of the therapeutic proteinand decreasing immunogenicity. See U.S. Pat. No. 4,179,337, Davis etal., issued Dec. 18, 1979. For a review, see Abuchowski et al., inEnzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds. pp. 367-383(1981)). A review article describing protein modification and fusionproteins is Francis, Focus on Growth Factors 3: 4-10 (May 1992)(published by Mediscript, Mountview Court, Friern Barnet Lane, LondonN20, 0LD, UK). For the present continuous administration, it ispreferred that the chemical modification allow for an increase incirculation time of the protein, so that a dosage of about 1 mg protein(exclusive of chemical moiety)/kg body weight of a mammal/day or lesswill result in weight loss of a mammal. The present continuousadministration will provide for weight loss of approximately 5% of bodymass in a period of 7 or fewer days.

[0011] The chemical moieties suitable for derivatization may be selectedfrom among water soluble polymers. The polymer selected should be watersoluble so that the protein to which it is attached does not precipitatein an aqueous environment, such as a physiological environment.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable. One skilled in the art willbe able to select the desired polymer based on such considerations aswhether the polymer/protein conjugate will be used therapeutically, andif so, the desired dosage, circulation time, resistance to proteolysis,and other considerations. For the present proteins and peptides, theeffectiveness of the derivatization may be ascertained by administeringthe derivative, in the desired form (i.e., by osmotic pump, or, morepreferably, by injection or infusion, or, further formulated for oral,pulmonary or nasal delivery, for example), and measuring weight loss.

[0012] The water soluble polymer may be selected from the groupconsisting of, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayhave advantages in manufacturing due to its stability in water.

[0013] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 2 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0014] The number of polymer molecules so attached may vary, and oneskilled in the art will be able to ascertain the effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

[0015] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art. E.g., EP 0 401384 herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylationof GM-CSF using tresyl chloride). For example, polyethylene glycol maybe covalently bound through amino acid residues via a reactive group,such as, a free amino or carboxyl group. Reactive groups are those towhich an activated polyethylene glycol molecule may be bound. The aminoacid residues having a free amino group may include lysine residues andthe N-terminal amino acid residue. Those having a free carboxyl groupmay include aspartic acid residues, glutamic acid residues, and theC-terminal amino acid residue. Sulfhydrl groups may also be used as areactive group for attaching the polyethylene glycol molecule(s).Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group. Attachment at residuesimportant for receptor binding should be avoided if receptor binding isdesired.

[0016] One may specifically desire N-terminally chemically modifiedprotein. Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pK_(a)differences between the ε-amino group of the lysine residues and that ofthe α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may be used.

[0017] In yet another aspect of the present invention, provided aremethods of using pharmaceutical compositions of the proteins andderivatives. Such pharmaceutical compositions may be for administrationfor injection, or for oral, pulmonary, nasal or other forms ofadministration which allow for the desired circulating dose of about 1mg protein/kg body weight/day or less. In general, comprehended by theinvention are pharmaceutical compositions comprising effective amountsof protein or derivative products of the invention together withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol); incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hylauronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the present proteins and derivatives. See,e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form. The effectiveamounts are those herein described.

[0018] The OB proteins and derivatives described are useful formodulation of the rate or quantity of fat cell deposition in a mammal.This is thought to be accomplished, in part, by a reduction in appetite,i.e., a reduction in food intake. Thus, one observable result is weightloss, or, put another way, a method of treating excess weight (viaweight loss). Thus, the present compositions are useful for themanufacture of a medicament for treating excess weight in a mammal.Another aspect is a method for reducing appetite. Either of theseaspects, modulation of fat deposition or modulation of appetite, areparticularly important treatments for humans (or other mammals) whodesire to lose weight.

[0019] One skilled in the art will be able to ascertain other effectivedosages by administration and observing weight loss. Here, the dosage of1 mg protein/kg body weight/day or less was seen to be particularlyeffective, when administered on a continuous basis. More particularly,the dosage of 0.5 mg/kg body weight/day was seen to be particularlyeffective on normal mice. Excess weight refers to body mass for whichremoval is desired. It is contemplated that the present compositions andmethods will be used to treat cases where removal of such excess weight(as a result of the present invention) will benefit other healthconcerns, such as diabetes, high blood pressure or cardiac problems,high cholesterol levels, low locomotion levels and other manifestationsof excess weight. As such, the present compositions and methods may beused in conjunction with other medicaments, such as those useful for thetreatment of diabetes (e.g., insulin, and possibly amylin), cholesteroland blood pressure lowering medicaments, and locomotion increasingmedicaments (e.g., amphetamines). Such administration may besimultaneous or may be in serriatim.

[0020] In addition, the present compositions and methods may be used inconjunction with surgical procedures, such as cosmetic surgeriesdesigned to alter the overall appearance of a body (e.g., liposuction orlaser surgeries designed to reduce body mass). The health benefits ofcardiac surgeries may be increased with concomitant use of the presentcompositions and methods.

[0021] Therefore, the present invention encompasses a method of treatingexcess weight in a mammal by continuous administration of 1 mgprotein/kg body weight/day or less of an OB protein selected from thegroup consisting of:

[0022] (a) recombinant methionyl murine OB protein (SEQ. ID. No. 2);

[0023] (b) recombinant methionyl human OB protein (SEQ ID No. 1);

[0024] (c) the protein of (a) or (b) lacking the methionyl residue atposition −1;

[0025] (d) the protein of (a), (b) or (c) lacking a glutamine atposition 28; and

[0026] (e) a chemically modified derivative of (a), (b),(c) or (d),wherein the chemical modification allows for an increase in circulationtime.

[0027] Preferably, the composition of subpart (e) is a pegylatedderivative, and, more preferably, an N-terminally pegylated derivative.

[0028] The derivative of subpart (e) allows for continuousadministration of the protein by increasing the circulation time of the(unmodified) protein. The present invention also encompasses a method oftreating excess weight where the method of continuous administration isby implantable pump, such as an osmotic pump.

[0029] In other aspects, the present invention relates to recombinantmurine and recombinant human OB DNAs and proteins, such as those of SEQ.ID NOs. 1, 2, 3, and 4, below. The recombinant proteins below arebacterially expressed, and contain N-terminal methionyl residues.Vectors and host cells useful for producing such proteins are alsoprovided. The vectors include pCFM1656 containing SEQ ID No. 1 or 3, andhost cells containing such vectors.

[0030] Methods for preparation of the recombinant proteins are alsoprovided, including methods for fermentation and methods forpurification.

[0031] In particular, the use of sarcosine for refolding of OB proteinin solution, obtained from bacterial inclusion bodies, provided forextremely efficient refolding. When proteins are expressed in bacteria,they may not be in the proper three-dimensional configuration, or, asreferred to herein, properly refolded. The three dimensionalconfiguration may be critical for biological activity, and storagestability. Although Sarckosyl has been used in processes forpurification of another protein (G-CSF, e.g., WO 89/10932),surprisingly, the use of sarcosine for the OB protein has resulted in arefolding efficiency of over 95%. Contemplated herein is the use ofN-lauroylsarcosine in a range of 0.5% -2.0% weight per volume of OBprotein in solution (obtained from inclusion bodies). With the use of 1%sodium sarcosine, the refolding efficiency, as determined by SDS PAGEand reverse phase HPLC, was 95% or greater. While one skilled in the artwill recognize that other compositions may be used for refolding, theuse of N-lauroyl sarcosine, as illustrated in the examples below, isparticularly advantageous for providing extremely efficient refolding.The removal of sarcosine was accomplished using Dowex®.

[0032] Therefore, the present invention also includes a method ofrefolding partially purified OB protein in a solution obtained frominclusion bodies, said partially purified OB protein selected from thegroup consisting of:

[0033] (a) recombinant methionyl murine OB protein (SEQ. ID. No. 2);

[0034] (b) recombinant methionyl human OB protein (SEQ ID No. 1);

[0035] (c) the protein of (a) or (b) lacking the methionyl residue atposition −1;

[0036] wherein said refolding is accomplished using sarcosine.

[0037] The present invention also includes methods of wherein saidN-lauroyl sarcosine is used at a concentration of 0.5% -2.0% weight pervolume of solution, and, more particularly, the use of 1% N-lauroylsarcosine. An oxidizing agent, such as copper sulfate, is also used inthe refolding process.

[0038] The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1 Use of Murine OB Protein in a Continuous Pump Infusion System

[0039] This example demonstrates that continuous infusion of OB proteinresults in weight loss in normal mice. Normal (non-obese) mice wereadministered murine OB protein via osmotic pump infusion. A dosage of0.5 mg protein/kg body weight/day resulted in a 4.62% (+/−1.34%) lossfrom baseline weight by the 6th day of infusion.

Materials and Methods

[0040] Animals

[0041] Wild type (+/+) C57B16 mice were used for this experiment. Theage of the mice at the initial time point was 8 weeks, and the animalswere weight stabilized. 10 mice were used for each cohort (vehicle vs.protein).

[0042] Animal Handling

[0043] Feeding and Weight Measurement

[0044] Mice were given ground rodent chow (PMI Feeds, Inc.) in powderedfood feeders (Allentown Caging and Equipment) which allowed a moreaccurate and sensitive measurement than use of regular block chow.Weight was measured at the same time each day (2:00 p.m.), for a periodof 6 days. Body weight on the day prior to the infusion was defined asbaseline weight. The mice used weighed 18-22 grams.

[0045] Housing

[0046] Mice were single-housed, and maintained under humane conditions.

[0047] Administration of Protein or Vehicle

[0048] Protein (as described below) or vehicle (phosphate bufferedsaline, pH 7.4) were administered by osmotic pump infusion. Alzetosmotic minipumps (Alza, Palo Alto, Calif., model no. 1007D) weresurgically placed in each mice in a subcutaneous pocket in thesubscapular area . The pumps were calibrated to administer 0.5 μlprotein in solution per hour for a dosage of 0.5 mg protein/kg bodyweight/day.

[0049] Controls

[0050] Control animals were those who had a Alzet osmotic minipumpinfusing phosphate buffered saline (pH 7.4).

[0051] Protein

[0052] Recombinant murine OB protein was used for the presentexperiments, generally at a concentration of about 0.9 mg/ml phosphatebuffered saline, pH 7.4. The amino acid sequence (and DNA sequence) usedwas the following: !Recombinant murine met OB (double stranded) DNA?!and amino acid sequence: (Seq. ID. Nos. 1 and 2)TCTAGATTTGAGTTTTAACTTTTAGAAGGAGGAATAACATATGG 9-+---------+---------+---------+---------+-- 68AGATCTAAACTCAAAATTGAAAATCTTCCTCCTTATTGTATACC                                        M  V - TACCGATCCAGAAAGT-------+-------- ATGGCTAGGTCTTTCA   P  I  Q  K  VTCAGGACGACACCAAAACCTTAATTAAAACGATCGTTACGCGTA 69-+---------+---------+---------+---------+-- 128AGTCCTGCTGTGGTTTTGGAATTAATTTTGCTAGCAATGCGCAT Q  D  D  T  K  T  L  I  K  T  I  V  T  R  I - TCAACGACATCAGTCA-------+-------- AGTTGCTGTAGTCAGT   N  D  I  S  HCACCCAGTCGGTCTCCGCTAAACAGCGTGTTACCGGTCTGGACT 129-+---------+---------+---------+---------+-- 188GTGGGTCAGCCAGAGGCGATTTGTCGCACAATGGCCAGACCTGA T  Q  S  V  S  A  K  Q  R  V  T  G  L  D  F - TCATCCCGGGTCTGCA-------+-------- AGTAGGGCCCAGACGT   I  P  G  L  HCCCGATCCTAAGCTTGTCCAAAATGGACCAGACCCTGGCTGTAT 189-+---------+---------+---------+---------+-- 248GGGCTAGGATTCGAACAGGTTTTACCTGGTCTGGGACCGACATA P  I  L  S  L  S  K  M  D  Q  T  L  A  V  Y - ACCAGCAGGTGTTAAC-------+-------- TGGTCGTCCACAATTG   Q  Q  V  L  TCTCCCTGCCGTCCCAGAACGTTCTTCAGATCGCTAACGACCTCG 249-+---------+---------+---------+---------+--GAGGGACGGCAGGGTCTTGCAAGAAGTCTAGCGATTGCTGGAGC 308 S  L  P  S  Q  N  V  L  Q  I  A  N  D  L  E - AGAACCTTCGCGACCT-------+-------- TCTTGGAAGCGCTGGA   N  L  R  D  LGCTGCACCTGCTGGCATTCTCCAAATCCTGCTCCCTGCCGCAGA 309-+---------+---------+---------+---------+-- 368CGACGTGGACGACCGTAAGAGGTTTAGGACGAGGGACGGCGTCT L  H  L  L  A  F  S  K  S  C  S  L  P  Q  T - CCTCAGGTCTTCAGAA-------+-------- GGAGTCCAGAAGTCTT   S  G  L  Q  KACCGGAATCCCTGGACGGGGTCCTGGAAGCATCCCTGTACAGCA 369-+---------+---------+---------+---------+-- 428TGGCCTTAGGGACCTGCCCCAGGACCTTCGTAGGGACATGTCGT P  E  S  L  D  G  V  L  E  A  S  L  Y  S  T - CCGAAGTTGTTGCTCT-------+-------- GGCTTCAACAACGAGA   E  V  V  A  LGTCCCGTCTGCAGGGTTCCCTTCAGGACATCCTTCAGCAGCTGGA 429-+---------+---------+---------+---------+-- 488CAGGGCAGACGTCCCAAGGGAAGTCCTGTAGGAAGTCGTCGACCT S  R  L  Q  G  S  L  Q  D  I  L  Q  Q  L  D -CGTTTCTCCGGAATGTTAATGGATCC 489 -------+---------+---------GCAAAGAGGCCTTACAATTACCTAGG   V  S  P  E  C

[0053] Herein, the first amino acid of the amino acid sequence forrecombinant protein is referred to as +1, and is valine, and the aminoacid at position −1 is methionine. The C-terminal amino acid is number146 (cysteine).

[0054] The cloning of the murine OB DNA for expression in E. coli wasdone as follows. The DNA sequence was deduced from the published peptidesequence that appeared in Zhang et al., Nature 372:425-432 (1994). Itwas reverse translated using E. coli optimal codons. The terminalcloning sites were XbaI to BamHI. A ribosomal binding enhancer and astrong ribosomal binding site were included in front of the codingregion. The duplex DNA sequence was synthesized using standardtechniques. Correct clones were confirmed by demonstrating expression ofthe recombinant protein and presence of the correct OB DNA sequence inthe resident plasmid.

[0055] Expression Vector and Host Strain

[0056] The plasmid expression vector used was pCFM1656, ATCC AccessionNo. 69576. The above DNA was ligated into the expression vector pCFM1656which had been linearized with XbaI and BamHI and transformed into theE. coli host strain, FM5. E. coli FM5 cells were derived at Amgen Inc.,Thousand Oaks, Calif. from E. coli K-12 strain (Bachmann, et al.,Bacteriol. Rev. 40: 116-167 (1976)) and contain the integrated lambdaphage repressor gene, cI₈₅₇ (Sussman et al., C.R. Acad. Sci. 254:1517-1579 (1962)). Vector production, cell transformation, and colonyselection were performed by standard methods. E.g., Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2d Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Host cells were grown in LBmedia.

[0057] Fermentation Process

[0058] A three-phase fermentation protocol was used known as a fed-batchprocess. Media compositions are set forth below.

[0059] Batch

[0060] A nitrogen and phosphate source were sterilized (by raising to122° C. for 35 minutes, 18-20 psi) in the fermentation vessel(Biolafitte, 12 liter capacity). Upon cooling, carbon, magnesium,vitamin, and trace metal sources were added aseptically. An overnightculture of the above recombinant murine protein-producing bacteria (16hours or more) of 500 mL (grown in LB broth) was added to the fermentor.

[0061] Feed I

[0062] Upon reaching between 4.0-6.0 OD₆₀₀, cultures were fed with FeedI. The glucose was fed at a limiting rate in order to control the growthrate (μ). An automated system (called the Distributive Control System)was instructed to control the growth rate to 0.15 generations per hour.

[0063] Feed II

[0064] When the OD600 had reached 30, culture temperature was slowlyincreased to 42° C. and the feed was changed to Feed II, below. Thefermentation was then allowed to continue for 10 hours with samplingevery 2 hours. After 10 hours, the contents of the fermentor was chilledto below 20° C. and harvested by centrifugation. Media Composition:Batch: 10 g/L Yeast extract 5.25 g/L (NH₄)₂SO₄ 3.5 g/L K₂HPO₄ 4.0 g/LKH₂PO₄ 5.0 g/L Glucose 1.0 g/L MgSO₄ · 7H₂O 2.0 mL/L Vitamin Solution2.0 mL/L Trace Metal Solution 1.0 mL/L P2000 Antifoam Feed I: 50 g/LBacto-tyrptone 50 g/L Yeast extract 450 g/L Glucose 8.75 g/L MgSO₄ ·7H₂O 10 mL/L Vitamin Solution 10 mL/L Trace Metal Solution Feed II: 200g/L Bacto-tryptone 100 g/L Yeast extract 110 g/L Glucose

[0065] Vitamin Solution (Batch and Feed I)

[0066] 0.5 g Biotin, 0.4 g Folic acid, and 4.2 g riboflavin, weredissolved in 450 mls H₂O and 3 mls 10 N NaOH, and brought to 500 mls inH₂O. 14 g pyridoxine-HCl and 61 g niacin were dissolved 150 ml H₂O and50 ml 10 N NaOH, and brought to 250 ml in H₂O. 54 g pantothenic acid wasdissolved in 200 ml H₂O, and brought to 250 ml. The three solutions werecombined and brought to 10 liters total volume.

[0067] Trace Metal Solution (Batch and Feed I)

[0068] Ferric Chloride (FeCl₃.6H₂O): 27 g/L

[0069] Zinc Chloride (ZnCl₂.4H₂O): 2 g/L

[0070] Cobalt Chloride (CoCl₂.6H₂O): 2 g/L

[0071] Sodium Molybdate (NaMoO₄.2H₂O): 2 g/L

[0072] Calcium Chloride (CaCl₂.2H₂O): 1 g/L

[0073] Cupric Sulfate (CuSO₄.5H₂O): 1.9 g/L

[0074] Boric Acid (H₃BO₃): 0.5 g/L

[0075] Manganese Chloride (MnCl₂.4H₂O): 1.6 g/L

[0076] Sodium Citrate dihydrate: 73.5 g/L

[0077] Purification Process for Murine OB Protein

[0078] Purification was accomplished by the following steps (unlessotherwise noted, the following steps were performed at 4° C.):

[0079] 1. Cell paste. E. coli cell paste was suspended in 5 times volumeof 7 mM of EDTA, pH 7.0. The cells in the EDTA were further broken bytwo passes through a microfluidizer. The broken cells were centrifugedat 4.2 K rpm for 1 hour in a Beckman J6-B centrifuge with a JS-4.2rotor.

[0080] 2. Inclusion body wash #1. The supernatant from above wasremoved, and the pellet was resuspended with 5 times volume of 7 mMEDTA, pH 7.0, and homogenized. This mixture was centrifuged as in step1.

[0081] 3. Inclusion body wash #2. The supernatant from above wasremoved, and the pellet was resuspended in ten times volume of 20 mMtris, pH 8.5, 10 mM DTT, and 1% deoxycholate, and homogenized. Thismixture was centrifuged as in step 1.

[0082] 4. Inclusion body wash #3. The supernatant from above was removedand the pellet was resuspended in ten times volume of distilled water,and homogenized. This mixture was centrifuged as in step 1.

[0083] 5. Refolding. The pellet was refolded with 15 volumes of 10 mMHEPES, pH 8.5, 1% sodium sarcosine (N-lauroyl sarcosine), at roomtemperature. After 60 minutes, the solution is made to be 60 μM coppersulfate, and then stirred overnight.

[0084] 6. Removal of sarcosine. The refolding mixture was diluted with 5volumes of 10 mM tris buffer, pH 7.5, and centrifuged as in step 1. Thesupernatant was collected, and mixed with agitation for one hour withDowex® 1-X4 resin (Dow Chemical Co., Midland Mich.), 20-50 mesh,chloride form, at 0.066% total volume of diluted refolding mix. See WO89/10932 at page 26 for more information on Dowex®. This mixture waspoured into a column and the eluant was collected. Removal of sarcosinewas ascertained by reverse phase HPLC.

[0085] 7. Acid precipitation. The eluant from the previous step wascollected, and pH adjusted to pH 5.5, and incubated for 30 minutes atroom temperature. This mixture was centrifuged as in step 1.

[0086] 8. Cation exchange chromatography. The pH of the supernatant fromthe previous step was adjusted to pH 4.2, and loaded on CM SepharoseFast Flow (at 7% volume). 20 column volumes of salt gradient were doneat 20 mM NaOAC, pH 4.2, 0 M to 1.0 M NaCl.

[0087] 9. Hydrophobic interaction chromatography. The CM Sepharose poolof peak fractions (ascertained from ultraviolet absorbance) from theabove step was made to be 0.2 M ammonium sulfate. A 20 column volumereverse salt gradient was done at 5 mM NaOAC, pH 4.2, with 0.4 M to 0 Mammonium sulfate. This material was concentrated and diafiltered intoPBS.

[0088] Results

[0089] Presented below are the percent (%) differences from baselineweight in C57B16J mice (8 weeks old):

[0090] Table 1: Weight Loss Upon Continuous Infusion Recombinant OB Time(days) Vehicle (PBS) protein Days 1-2 3.24 +/− 1.13  1.68 +/− 1.4 Days3-4 4.3 +/− .97 −2.12 +/− .79 Days 5-6 4.64 +/− .96  −4.62 +/− 1.3

[0091] As can be seen, at the end of a 6 day continuous infusion regime,animals receiving the OB protein lost over 4% of their body weight, ascompared to baseline. This is a substantially more rapid weight lossthan has been observed with intraperitoneal (i.p.) injection. Weightloss at the end of a 32-day injection period, in wild type (normal)mice, with daily i.p. injections of recombinant murine OB protein at a10 mg/kg dose was 2.6%, and had not been more than 4% at any time duringthe dosing schedule (data not shown). The present data indicate thatwith continuous infusion, a 20-fold lower dosage (0.5 mg/kg vs. 10mg/kg) achieves more weight loss in a shorter time period.

[0092] The results seen here are statistically significant, e.g., −4.62%with p<0.0001.

EXAMPLE 2 Dose Response Studies

[0093] An additional study demonstrated that there was a dose responseto continuous administration of OB protein. In this study, non-obese,CD-1 mice, weighing 35-40 g were administered recombinant murine OBprotein using methods similar to the above example. The results are setforth in Table 2, below, (with % body weight lost as compared tobaseline, measured as above):

Table 2: Dose Response with Continuous Administration

[0094] % Reduction in Dose Time body weight 0.03 Day 2 3.5 mg/kg/day 1mg/kg/day Day 2 7.5 1 mg/kg/day Day 4 14

[0095] As can be seen, increasing the dose from 0.03 mg/kg/day to 1mg/kg/day increased the weight lost from 3.5% to 7.5%. It is alsonoteworthy that at day 4, the 1 mg/kg/day dosage resulted in a 14%reduction in body weight.

EXAMPLE 3 Cloning and Expression of a Recombinant Human Methionyl OBProtein

[0096] This example provides compositions and methods for preparation ofa recombinant human version of the OB protein.

[0097] The human version of the OB DNA was constructed from the murineOB DNA, as in Example 1, above, by replacing the region between the MluIand BamHI sites with duplex DNA (made from synthetic oligonucleotides)in which 20 codon substitutions had been designed. The MluI site isshown under the solid line in the sequence below. This DNA was put intothe pCFM1656 vector (ATCC Accession No. 69576), in the same fashion asthe recombinant murine protein, as described above. Herein, the firstamino acid of the amino acid sequence for recombinant human proteinbelow is referred to as +1, and is valine, and the amino acid atposition −1 is methionine. The C-terminal amino acid is number 146(cysteine). !Recombinant murine met OB (double stranded) DNA? !and AMINOacid sequence: (Seq. ID. Nos. 3 and 4)CATATGGTACCGATCCAGAAAGTTCAGGACGACACCAAAACCTT 1---------+---------+---------+---------+---- 60GTATACCATGGCTAGGTCTTTCAAGTCCTGCTGTGGTTTTGGAA   M  V  P  I  Q  K  V  Q  D  D  T  K  T  L - AATTAAAACGATCGTT-----+---------+ TTAATTTTGCTAGCAA  I  K  T  I  VACGCGTATCAACGACATCAGTCACACCCAGTCGGTGAGCTCTAA 61---------+---------+---------+---------+---- 120TGCGCATAGTTGCTGTAGTCAGTGTGGGTCAGCCACTCGAGATTT  R  I  N  D  I  S  H  T  Q  S  V  S  S  K - ACAGCGTGTTACAGGC-----+---------+ TGTCGCACAATGTCCG  Q  R  V  T  GCTGGACTTCATCCCGGGTCTGCACCCGATCCTGACCTTGTCCAAA 121---------+---------+---------+---------+---- 180GACCTGAAGTAGGGCCCAGACGTGGGCTAGGACTGGAACAGGTTTL  D  F  I  P  G  L  H  P  I  L  T  L  S  K - ATGGACCAGACCCTG-----+---------+ TACCTGGTCTGGGAC  M  D  Q  T  LGCTGTATACCAGCAGATCTTAACCTCCATGCCGTCCCGTAACGT 181---------+---------+---------+---------+---- 240CGACATATGGTCGTCTAGAATTGGAGGTACGGCAGGGCATTGCAA  V  Y  Q  Q  I  L  T  S  M  P  S  R  N  V - TCTTCAGATCTCTAAC-----+---------+ AGAAGTCTAGAGATTG  L  Q  I  S  NGACCTCGAGAACCTTCGCGACCTGCTGCACGTGCTGGCATTCTC 241---------+---------+---------+---------+---- 300CTGGAGCTCTTGGAAGCGCTGGACGACGTGCACGACCGTAAGAGD  L  E  N  L  R  D  L  L  H  V  L  A  F  S - CAAATCCTGCCACCTG-----+---------+ GTTTAGGACGGTGGAC  K  S  C  H  LCCATGGGCTTCAGGTCTTGAGACTCTGGACTCTCTGGGCGGGGT 301---------+---------+---------+---------+---- 360GGTACCCGAAGTCCAGAACTCTGAGACCTGAGAGACCCGCCCCAP  W  A  S  G  L  E  T  L  D  S  L  G  G  V - CCTGGAAGCATCCGGT-----+---------+ GGACCTTCGTAGGCCA  L  E  A  S  GTACAGCACCGAAGTTGTTGCTCTGTCCCGTCTGCAGGGTTCCCT 361---------+---------+---------+---------+---- 420ATGTCGTGGCTTCAACAACGAGACAGGGCAGACGTCCCAAGGGAY  S  T  E  V  V  A  L  S  R  L  Q  G  S  L -TCAGGACATGCTTTGGGAGCTGGACCTGTCTCCGGGTTGTTAAT 421-----+---------+---------+---------+-------- 454AGTCCTGTACGAAACCGTCGACCTGGACAGAGGCCCAACAATTA Q  D  M  L  W  Q  L  D  L  S  P  G  C  * GGATCC -+---- CCTAGG

[0098] Fermentation

[0099] Fermentation of the above host cells to produce recombinant humanOB protein was accomplished using the conditions and compositions asdescribed above for recombinant murine material. The results wereanalyzed for yield (grams ob DNA product/liter of fermentation broth),prior to purification of the recombinant human OB material. (Minoramounts of bacterial protein were present.) Bacterial expression wasalso calculated. TABLE 3 Analysis of Human OB Protein Expression ODYield Expression Timepoint (@ 600 nm) (g/L) (mg/OD · L) Ind. + 2 47 1.9141 hours. Ind. + 4 79 9.48 120 hours. Ind. + 6 95 13.01 137 hours.Ind. + 8 94 13.24 141 hours. Ind. + 10 98 14.65 149 hours.

[0100] abbreviations: Ind. +______ hours means the hours after inductionof protein expression, as described in Example I for the recombinantmurine material using pCFM1656

[0101] OD: optical density, as measured by spectrophotometer 20milligrams per OD unit per liter

[0102] mg/OD-L: expression in terms of milligrams of protein per OD unitper liter.

[0103] g/L: grams protein/liter fermentation broth

[0104] Purification of the Recombinant Human OB Protein

[0105] Recombinant human protein may be purified using methods similarto those used for purification of recombinant murine protein, as inExample 1, above. For preparation of recombinant human OB protein, step8 was performed by adjusting the pH of the supernatant from step 7 to pH5.0, and loading this onto a CM Sepharose fast flow column. The 20column volume salt gradient was performed at 20 mM NaOAC, pH 5.5, 0M to0.5 M NaCl. Step 9 was performed by diluting the CM Sepharose pool fourfold with water, and adjusting the pH to 7.5. This mixture was made to0.7 M ammonium sulfate. Twenty column volume reverse salt gradient wasdone at 5 mM NaOAC, pH 5.5, 0.2 M to 0 M ammonium sulfate. Otherwise,the above steps were identical.

[0106] While the present invention has been described in terms ofpreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

1 6 491 base pairs nucleic acid double linear cDNA 1 TCTAGATTTGAGTTTTAACT TTTAGAAGGA GGAATAACAT ATGGTACCGA TCCAGAAAGT 60 TCAGGACGACACCAAAACCT TAATTAAAAC GATCGTTACG CGTATCAACG ACATCAGTCA 120 CACCCAGTCGGTCTCCGCTA AACAGCGTGT TACCGGTCTG GACTTCATCC CGGGTCTGCA 180 CCCGATCCTAAGCTTGTCCA AAATGGACCA GACCCTGGCT GTATACCAGC AGGTGTTAAC 240 CTCCCTGCCGTCCCAGAACG TTCTTCAGAT CGCTAACGAC CTCGAGAACC TTCGCGACCT 300 GCTGCACCTGCTGGCATTCT CCAAATCCTG CTCCCTGCCG CAGACCTCAG GTCTTCAGAA 360 ACCGGAATCCCTGGACGGGG TCCTGGAAGC ATCCCTGTAC AGCACCGAAG TTGTTGCTCT 420 GTCCCGTCTGCAGGGTTCCC TTCAGGACAT CCTTCAGCAG CTGGACGTTT CTCCGGAATG 480 TTAATGGATC C491 491 base pairs nucleic acid double linear cDNA 2 AGATCTAAACTCAAAATTGA AAATCTTCCT CCTTATTGTA TACCATGGCT AGGTCTTTCA 60 AGTCCTGCTGTGGTTTTGGA ATTAATTTTG CTAGCAATGC GCATAGTTGC TGTAGTCAGT 120 GTGGGTCAGCCAGAGGCGAT TTGTCGCACA ATGGCCAGAC CTGAAGTAGG GCCCAGACGT 180 GGGCTAGGATTCGAACAGGT TTTACCTGGT CTGGGACCGA CATATGGTCG TCCACAATTG 240 GAGGGACGGCAGGGTCTTGC AAGAAGTCTA GCGATTGCTG GAGCTCTTGG AAGCGCTGGA 300 CGACGTGGACGACCGTAAGA GGTTTAGGAC GAGGGACGGC GTCTGGAGTC CAGAAGTCTT 360 TGGCCTTAGGGACCTGCCCC AGGACCTTCG TAGGGACATG TCGTGGCTTC AACAACGAGA 420 CAGGGCAGACGTCCCAAGGG AAGTCCTGTA GGAAGTCGTC GACCTGCAAA GAGGCCTTAC 480 AATTACCTAG G491 147 amino acids amino acid single linear protein 3 Met Val Pro IleGln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys 1 5 10 15 Thr Ile ValThr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser 20 25 30 Ala Lys GlnArg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro 35 40 45 Ile Leu SerLeu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln 50 55 60 Val Leu ThrSer Leu Pro Ser Gln Asn Val Leu Gln Ile Ala Asn Asp 65 70 75 80 Leu GluAsn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser 85 90 95 Cys SerLeu Pro Gln Thr Ser Gly Leu Gln Lys Pro Glu Ser Leu Asp 100 105 110 GlyVal Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser 115 120 125Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Val Ser 130 135140 Pro Glu Cys 145 454 base pairs nucleic acid double linear cDNA 4CATATGGTAC CGATCCAGAA AGTTCAGGAC GACACCAAAA CCTTAATTAA AACGATCGTT 60ACGCGTATCA ACGACATCAG TCACACCCAG TCGGTGAGCT CTAAACAGCG TGTTACAGGC 120CTGGACTTCA TCCCGGGTCT GCACCCGATC CTGACCTTGT CCAAAATGGA CCAGACCCTG 180GCTGTATACC AGCAGATCTT AACCTCCATG CCGTCCCGTA ACGTTCTTCA GATCTCTAAC 240GACCTCGAGA ACCTTCGCGA CCTGCTGCAC GTGCTGGCAT TCTCCAAATC CTGCCACCTG 300CCATGGGCTT CAGGTCTTGA GACTCTGGAC TCTCTGGGCG GGGTCCTGGA AGCATCCGGT 360TACAGCACCG AAGTTGTTGC TCTGTCCCGT CTGCAGGGTT CCCTTCAGGA CATGCTTTGG 420CAGCTGGACC TGTCTCCGGG TTGTTAATGG ATCC 454 454 base pairs nucleic aciddouble linear cDNA 5 GTATACCATG GCTAGGTCTT TCAAGTCCTG CTGTGGTTTTGGAATTAATT TTGCTAGCAA 60 TGCGCATAGT TGCTGTAGTC AGTGTGGGTC AGCCACTCGAGATTTGTCGC ACAATGTCCG 120 GACCTGAAGT AGGGCCCAGA CGTGGGCTAG GACTGGAACAGGTTTTACCT GGTCTGGGAC 180 CGACATATGG TCGTCTAGAA TTGGAGGTAC GGCAGGGCATTGCAAGAAGT CTAGAGATTG 240 CTGGAGCTCT TGGAAGCGCT GGACGACGTG CACGACCGTAAGAGGTTTAG GACGGTGGAC 300 GGTACCCGAA GTCCAGAACT CTGAGACCTG AGAGACCCGCCCCAGGACCT TCGTAGGCCA 360 ATGTCGTGGC TTCAACAACG AGACAGGGCA GACGTCCCAAGGGAAGTCCT GTACGAAACC 420 GTCGACCTGG ACAGAGGCCC AACAATTACC TAGG 454 147amino acids amino acid single linear protein 6 Met Val Pro Ile Gln LysVal Gln Asp Asp Thr Lys Thr Leu Ile Lys 1 5 10 15 Thr Ile Val Thr ArgIle Asn Asp Ile Ser His Thr Gln Ser Val Ser 20 25 30 Ser Lys Gln Arg ValThr Gly Leu Asp Phe Ile Pro Gly Leu His Pro 35 40 45 Ile Leu Thr Leu SerLys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln 50 55 60 Ile Leu Thr Ser MetPro Ser Arg Asn Val Leu Gln Ile Ser Asn Asp 65 70 75 80 Leu Glu Asn LeuArg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser 85 90 95 Cys His Leu ProTrp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly 100 105 110 Gly Val LeuGlu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser 115 120 125 Arg LeuGln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser 130 135 140 ProGly Cys 145

1. A method of treating excess weight in a mammal by continuousadministration of 1 mg protein/kg body weight/day or less of an OBprotein selected from the group consisting of: (a) recombinant methionylmurine OB protein (SEQ. ID. No. 2); (b) recombinant methionyl human OBprotein (SEQ ID No. 1); (c) the protein of (a) or (b) lacking themethionyl residue at position −1; (d) the protein of (a), (b) or (c)lacking a glutamine at position 28; and (e) a chemically modifiedderivative of (a), (b),(c) or (d).
 2. A method of claim 1 wherein thechemically modified derivative is a pegylated derivative.
 3. A method ofclaim 2 wherein the pegylated derivative is N-terminally pegylated.
 4. Amethod of claim 1 wherein said continuous administration is accomplishedby osmotic pump.
 5. A DNA sequence according to SEQ ID No.
 1. 6. Avector containing a DNA sequence according to claim
 5. 7. A vector ofclaim 6 wherein said vector is pCFM1656.
 8. A DNA sequence according toSEQ ID No.
 3. 9. A vector containing a DNA sequence according to claim8.
 10. A vector according to claim 9 wherein said vector is pCFM1656.11. A method of refolding partially purified OB protein in a solutionobtained from inclusion bodies, said partially purified OB proteinselected from the group consisting of: (a) recombinant methionyl murineOB protein (SEQ. ID. No. 2); (b) recombinant methionyl human OB protein(SEQ ID No. 1); (c) the protein of (a) or (b) lacking the methionylresidue at position −1; wherein said refolding is accomplished usingN-lauroyl sarcosine.
 12. A method of claim 11 wherein said sarcosine isused at a concentration of 0.5%-2.0% weight per volume of solution.